The long term objective of this project is to understand the function and regulation of protein kinases and phosphatases in control of cell growth and differentiation, and the molecular mechanism of growth factor induced signal transduction. The mitogen-activated protein kinases (MAPK), also known as the extracellular signal-regulated kinases (ERK), are acutely activated by numerous extracellular signals. These stimuli include mitogenic growth factors (such as insulin, epidermal growth factor, platelet derived growth factor, fibroblast growth factor, and nerve growth factor), cytokines, T-cell antigens, and phorbol esters. Accumulating evidence supports the hypothesis that activation of ERK plays an essential role in these signal transduction events. The direct upstream activator of ERK is a dual-specific kinase, MEK, which phosphorylates ERK on threonine and tyrosine residues. MEK acts as an integration point for at least two distinct signal transduction pathways; one is initiated by receptor tyrosine kinase whereas the other utilizes by receptors coupled to trimeric G-protein. Thus, research into the regulation of ERK is now more appropriately focused on the regulation of MEK. The first specific aim of this proposal is to study the molecular mechanism of MEK activation by identifying the regulatory phosphorylation sites. Physiological importance of MEK phosphorylation and activation will be tested in Saccharomyces cerevisiae. MEK is a family of kinases whose activation has been observed in many biological processes. New members of the MEK family will be isolated and their role in various signal transduction events will be investigated. Data from our laboratory have shown that MEKA (for MEK activator) is the major MEK activator responsible for growth factor induced MEK activation. Experiments are designed to purify and characterize MEKA, and finally to isolate cDNA of MEKA. A mitogen induced dual specific phosphatase has been isolated and shown to specifically dephosphorylate and in activate ERK in vitro. To obtain in vivo evidence for the regulation of ERK by dephosphorylation, we plan to study roles of the dual specific phosphatase on ERK function in cultured cells. Biochemical, molecular biological immunological, cell biological and genetic techniques will be used to accomplish specific aims outlined in this proposal. Data generated through the course of this project will shed light on the regulatory mechanism of the ERK/MEK kinase cascade.